Biochemistry 1983, 22, 3448-3452
3448
Steric Course of Hydrogen Transfer during Enzymatic Formation of 3a-Heteroyohimbine Alkaloidst Joachim Stockigt,* Thomas Hemscheidt, Gerd Hofle,* Peter Heinstein.5 and Victor Fornacekil
ABSTRACT:
With a crude enzyme extract obtained from Catharanthus roseus cell-suspension cultures, deuterium has been used to follow the cell-free biosynthesis of 3a-heteroyohimbine alkaloids. In the presence of D 2 0 one D+ is incorporated into the C-19 methyl group and the other D+ into the C-20 position of the heteroyohimbines ajmalicine, 19-epiajmalicine, and tetrahydroalstonine, respectively. During the final reduction
step, cathenamine reductase(s) stereospecifically transfer(s) D- from NADPD into the C-21a position of the three ajmalicine isomers. Formation of these 3a-heteroyohimbines, starting from geissoscliizine as a precursor, proceeds by channeling the precursor into the main pathway by a stereospecific dehydrogenation. Deuterium incorporation was established by spectroscopic methods.
x e overall biosynthetic sequence leading to 3a-heteroyohimbine alkaloids has been extensively studied over the past 6 years with enzymes isolated from cell-suspension cultures of Catharanthus roseus (Zenk, 1980, and references cited therein; Stiickigt, 1980, and references cited therein). So far, most of the biogenetic intermediates of the major pathway (tryptamine secologanin 3a-heteroyohimbines) or of the side reaction leading to the major pathway (geissoschizine 4,21-dehydrogeissoschizine 3a-heteroyohimbines) have already been identified (Figure 1). Moreover, the enzymes involved are known and most have been characterized in detail (Treimer & Zenk, 1979; Hemscheidt & Zenk, 1980; Pfitzner & Stockigt, 1982). However, several questions concerning some outstanding mechanistic problems still need to be answered; for instance, the extent of incorporation of H+ during the intermediary formation of the key compound cathenamine and the stereochemistry of the NADPH-dependent reduction of this intermediate are entirely unknown. A study of these aspects, therefore, appeared to be necessary. After the complete 'HNMR' analyses had been performed for ajmalicine, its C- 19 epimer, tetrahydroalstonine, and geissoschizine (Hofle et al., 1980; Lounasmaa & Kan, 1980), an analytical method was thus available to investigate the steric mode of deuterium incorporation during the cell-free biosynthesis of the three ajmalicine isomers. Here, we report that, during the enzymatic formation of cathenamine, one proton is incorporated into the C-19 methyl group. In the subsequent reduction step, a further proton ends up at C-20 while the hydride ion of NADPH is stereospecifically transferred into the C-2 1 position, furnishing the three heteroyohimbine alkaloids, ajmalicine, 19-epiajmalicine, and tetrahydroalstonine. For the sequence geissoschizine 3a-heteroyohimbine alkaloids, the stereochemistry of the involved reduction step was
found to be the same. This fact also supports the finding (Pfitzner & Stijckigt, 1982) that geissoschizine is biogenetically converted into 4,2 1-dehydrogeissoschizine by a dehydrogenation step that leads to the main pathway of heteroyohimbine synthesis.
+
-
-
-
-
From the Department of Pharmazeutische Biologie, Universitat Munchen, 8000 Miinchen 2, FRG. Received December IO, 1982. This research was financially supported by the Deutsche Forschungsgemeinschaft (SFB 145). Part of this work was presented by J.S. in lectures held at the Phytochemical Society Symposium on Indole and Biogenetically Related Alkaloids, London, 1979, and at the 28th IUPAC Congress, Vancouver, 198 1. *Present address: Gesellschaft fur Biotechnologische Forschung, 3300 Braunschweig, FRG. s Present address: Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907. 11 Present address: Bruker Analytische Messtechnik GmbH, 7512 Rheinstetten-Fo., FRG.
0006-2960/83/0422-3448$01.50/0
Materials and Methods Chemicals and Biochemicals. Dowex AG 1-X4 was obtained from Bio-Rad; poly(vinylpyrro1idone) was from Fa. Heidinger. Deuterated water (99.7% atom % D) was from Roth and EGA-Chemie; [2H,]ethanol (99%) was purchased from Merck; deuterated chloroform (99.96%) for NMR analyses was obtained from Merck and from Merck Sharp & Dohme, respectively. Pyridine nucleotides, alcohol dehydrogenase from Leuconostoc mesenteroides (EC l. l . l .2), and isocitrate dehydrogenase from pig heart (EC 1.l. 1.42) were from Boehringer. Strictosidine was a gift of U. Pfitzner from our laboratory. Cell Cultures and Enzyme Preparation. Suspension cultures of Catharanthus roseus were grown for 8 days in a standard production medium (Zenk et al., 1977). Cells to be used as an enzyme source were harvested, immediately frozen with liquid nitrogen, and submitted to the following procedure: 160 g of frozen cells was crushed with an IKA Universalmuhle M 20 in the presence of 110 g of PVP (prewashed with 50 mM phosphate buffer, pH 7.0). The mixture was stirred at 0 "C for 60 min in 500 mL of 50 mM phosphate buffer (pH 7.0, containing 10 NL of /3-mercaptoethanol). After the resultant slurry was passed through cheesecloth and centrifuged at 27300g for 10 min, the protein in the supernatant was precipitated with 30-70% (NH4)2S04(w/v). The residue was then redissolved in 17 mL of the above buffer (without pmercaptoethanol). This solution was stirred for 15 min at 0 "C with 3 g of the anion-exchange resin Dowex AG 1-X4, 100-200 mesh, borate form. The centrifuge filtrate obtained was freed of endogenous alkaloids and cofactors by applying the solution to a Sephadex G-25 column (2.5 X 30 cm, flow rate 0.5 mL/min). The resulting enzyme solution was used in the experiments described below. For study of the incorporation of solvent protons (deuterium), the desalted protein solution was concentrated by further precipitation with (NHJ2S04(30-70%) and the enzyme exhaustively dialyzed
'
Abbreviations: PVP, poly(viny1pyrrolidone); NMR, nuclear magnetic resonance; TLC, thin-layer chromatography; MS, mass spectroscopy; Tris, tris(hydroxymethy1)aminomethane.
0 1983 American Chemical Society
HETEROYOHIMBINE BIOSYNTHESIS:
HYDROGEN TRANSFER
VOL. 22, NO. 14, 1983
Strictosidine aglycone
St r ictosidine
-
-
c
Dialdehyde
OH L,Zl-Dehydrocorynan theine aldehyde
Carbinolamine
-
c.
20,Zl- Didehydrocorynan theine aldehyde
-
OH
421- Dehydrogeisso -
Cat henamine
lminium cat henamtne
NADPD NADP@
NADP@ NADPD
H H" H*' Me02C
YD
schizine
19-epi- Cat henamine
Geissoschizine
CH2D
CH2D 19
\ o
19- rpi- Ajmalicine FIGURE
3449
19-epi- Cathenamine lminium form
AJmal icine Tet r a hyd ro a Is ionIne
1: Hydrogen transfer during cell-free biosynthesis of 3a-heteroyohimbine alkaloids as demonstrated by deuterium incorporation.
against deuterated buffer. Protein concentrations were determined as previously described (Bradford, 1976). Purification and Spectroscopic Analyses of Enzymatically Formed Products. Purification of cell free formed alkaloids was carried out by TLC chromatography on prewashed (methanol) silica gel plates (Polygram Si1 G/UVzS4;Machery-Nagel) in the following solvent systems: (A) acetonepetroleum ether (40-60 OC)-diethylamine, 2:7: 1; (B) ethyl acetate-ether-n-hexane, 2:2: 1; (C) chloroform-methanolammonia, 9O:lO:O.l. All solvents were of the highest purity commercially available. Yields of the synthesized alkaloids were determined by UV spectrophotometry. The structural determination of the products was based on UV, MS, and * HN M R data. UV spectra were obtained on
a Perkin-Elmer 551 S spectrophotometer in methanol (Uvasol) as solvent; mass spectra (MS) and deuterium incorporation were measured on a Finnigan MAT 44 S at 70 eV (E1 mode). The 'H N M R spectra were obtained with a Bruker WH 400 or WM 500 FT spectrometer in CDC1, or [2H6]acetone, using 30-1000 pg of sample in 0.4 mL of solvent. Depending on the isolated amounts of alkaloids, 500-19 000 transients were accumulated in a 32K memory. Results Cell-Free Formation of 3a-Heteroyohimbine Alkaloids. For the synthesis of [ 18-2H]cathenamine (analyzed as its BH4 reduction product tetrahydroalstonine), 8.4 mg (42.8 pmol) of tryptamine hydrochloride was incubated with 78.6 mg (202
3450
STOCKIGT ET A L .
BIOCHEMISTRY
pmol) of secologanin in the presence of 0.1 M phosphate buffer (pH 7.5) and 35.3 mg of protein in a total volume of 45 mL of D 2 0 for 120 min at 30 "C. For the reduction of the enzymatically formed cathenamine to tetrahydroalstonine (Husson et al., 1977), an excess of NaBH, and 2 mL of [aH]methanol were added. After 40 min, the mixture was extracted with ether. The organic layer was washed with water and concentrated, and the residue was purified by TLC (systems A, B, and C ) , yielding tetrahydroalstonine (5 pmol). Tetrahydro[ 18,20-2H,]alstonine: mass spectrum, m l z 354 (M', 96), 353 (M' - H, loo), 339 (M' - CH,, 29), 225 (26); 'H NMR 4.48 (t, J = 6.5 Hz, 19-H) ppm. [ 18,20-2H2]-3a-Heteroyohimbine Alkaloids. For the enzymatic synthesis of 3a-heteroyohimbine alkaloids in deuterated medium, 22.5 mg (42.4 pmol) of strictosidine was allowed to react under NADPH-regenerating conditions in the presence of 124 mg (157 pmol) of NADP+Na2, 228 mg (880 pmol) of DL-isocitrate, 15 mg (750 nkat) of isocitrate dehydrogenase, 35.6 mg (373 pmol) of magnesium chloride, and 180 mg of C.roseus protein in 53 mL of deuterated phosphate buffer (50 mM, pH 7.5, 30 "C). After an incubation time of 120 min, the alkaloids produced were extracted with ethyl acetate at pH 7.5 and 8.5 (adjusted with NaHC03), respectively. The combined extracts were washed with water and concentrated, and the residue was twice chromatographed on TLC with systems A and B, yielding the deuterated alkaloids ajmalicine (1.6 pmol), 19-epiajmalicine (1.7 pmol), and tetrahydroalstonine (4.8 pmol). [ 18,20-2H,]Ajmalicine: mass spectrum, m l z 354 (M', 87), 353 (M' - H, 87), 339 (M' - CH3, 5); 'H N M R (['HJacetone) 2.27 (d, J = 11 Hz, 21a-H), 2.41 (dd, J = 12 Hz, J = 2 Hz, 15-H), 3.00 (d, J = 11 Hz, 21/3-H), 4.49 (t, J = 7 Hz, 19-H) ppm. [ 18,20-2H,]- 19-Epiajmalicine: mass spectrum, m l z 354 (M', loo), 353 (M' - H, 86), 339 (M' - CH3, 3); 'H NMR (CDClJ 2.23 (d, J = 11 Hz, 2la-H), 2.39 (br d, J = 11 Hz, 15-H), 3.89 (t, J = 6.5 Hz, 19-H) ppm. Tetrahydro[ 18,20-2H2]alstonine: mass spectrum, m l z 354 (M', 96), 353 (M' - H, loo), 339 (M' - CH,, 29), 225 (26); 'H NMR (CDC13) 2.73 (d, J = 12.5 Hz, 21a-H), 2.76 (dd, J = 4.5 Hz, J = 12 Hz, 15-H), 4.48 (t, J = 6.5 Hz, 19-H) PPm. [21a-2H]-3a-Heteroyohimbine Alkaloids. (a) Starting from strictosidine as precursor, in a total volume of 350 mL (1:l mixture of 75 mM borate and 75 mM Tris buffer, pH 7.5), 40 mg (75.3 pmol) of strictosidine, 285 mg (361 pmol) of NADP+Na2, 5.9 mg (3000 nkat) of alcohol dehydrogenase (from Leuconostoc mesenteroides), 2.85 mL of [2H6]ethanol, and 171 mg of protein were incubated at 30 "C for 120 min. Under these NADPD-regenerating conditions, the three ajmalicine isomers were synthesized and isolated as described above. After repeated purification (3 times) on TLC (systems A and B), 3.0 pmol of ajmalicine, 3.3 pmol of 19-epiajmalicine, and 5.9 pmol of tetrahydroalstonine were finally obtained and analyzed for their deuterium content and for the location of the incorporated deuterium by MS and 'H NMR. [21a-2H]Ajmalicine: mass spectrum, m l z 353 (M', 93), 352 (M' - H, loo), 338 (M' - CH3,6), 185 (35); 'H NMR (CDC13) 2.17 (br d, J = 11 Hz, 20-H), 2.45 (t, J = 12 Hz, 15-H), 2.98 (br s, 210-H) ppm. [21a-2H]-19-Epiajmalicine: mass spectrum m l z 353 (M', 95), 352 (M+ - H, loo), 338 (M' - CH,, 5), 185 (33); 'H NMR (CDC13) 2.40 (t, J = 11 Hz, 15-H) ppm (2la-H disappeared). Tetrahydro[2la2H]alstonine: mass spectrum, m / z 353 (M+, 80), 352 (M' - H, loo), 338 (M+- CH,, 25); 'H NMR ([2H,]acetone) 3.18 (br s, 210-H) ppm.
(b) Starting from geissoschizine as precursor, under NADPD-regenerating conditions, as described for (a), the sequence geissoschizine heteroyohimbines was investigated. The reaction mixture contained, in 43 mL, 70 mM phosphate buffer (pH 7.5), 4.5 mg (12.8 pmol) of geissoschizine, 78 mg (99 pmol) of NADP'Na2, 2.6 mL of [ZH,]ethanol,0.3 mg (1 53 nkat) of alcohol dehydrogenase from L. mesenteroides, and 94 mg of protein from C. roseus cell cultures. The incubation was carried out at 30 "C for 120 min, and the alkaloids formed were isolated and purified as described above. MS and 'H NMR data for the three isomers were identical with those obtained by the procedure as described for (a).
-
Discussion A biogenetic pathway can be elucidated by the cell-free investigation of single-reaction steps involved in the whole sequence. With this methodology, the enzymatic synthesis of the corresponding biogenetic intermediates can usually be achieved, and structural determination will provide information on the mechanistic aspects of a specific biogenetic step. However, this approach requires the usually tedious purification of a number of enzymes. An alternative approach is to use stable isotopes and spectral analysis of the synthesized intermediates and final products to elucidate the mechanisms and the stereochemistry of the majority of steps in a biosynthetic sequence. This allows the use of crude enzyme mixtures or cell-free homogenates that are incubated with and without cofactors and with the appropriate substrates, Le., intermediates in the pathway. We, therefore, used deuterium, in the form of D20, in the medium and deuterated cofactors for a more detailed understanding of the biogenetic route leading to monoterpenoid indole alkaloids of the 3a-heteroyohimbine type. By use of a crude enzyme extract from C. roseus cell-suspension cultures, the incorporation of D' and D- was investigated by MS and 'H NMR for the enzymatic conversion of strictosidine and geissoschizine into ajmalicine, 19-epiajmalicine and tetrahydroalstonine, the end products of the heteroyohimbine pathway. During the course of the reactions in the main pathway, the vinyl function of strictosidine is converted into a methyl group. Pathway intermediates exhibiting this methyl group, e.g., 4,21-dehydrogeissoschizine and the cathenamine isomers, are enzymatically formed in the absence of any cofactors. This indicates a nonreductive sequence, in which a proton of the ambient water might be added to the vinyl function. To clarify this assumption, the cell-free synthesis of cathenamine was carried out in D 2 0 instead of HzO, starting with tryptamine and secologanin and using the crude enzyme preparation. To stabilize the rather reactive intermediate, cathenamine, its enamine double bond was immediately reduced by BH4- after the incubation and the resulting stable tetrahydroalstonine analyzed by MS and IH NMR. The mass spectrum of the tetrahydroalstonine so produced showed the incorporation of two deuterium atoms by the increase of the mass ions m / z 352, 351, 337, and 223 to m l z 354, 353, 339, and 225, respectively. For the C-19 proton, a clear triplet was observed in the NMR spectrum at 4.48 ppm, demonstrating the quantitative incorporation of one D' into the C-19 methyl group and, during the chemical reduction step, into the (2-20 position. Therefore, during the biogenetic reactions of the main pathway, I>+ is incorporated at the stage of 20,21-dehydrocorynantheinealdehyde with the concomitant formation of 4,21-dehydrogeissoschizine. The final steps in the pathway are the NADPH-dependent reductions of cathenamine and its C-19 epimer. Investigation of this part of the sequence, using the experimental conditions
HETEROYOHIMBINE BIOSYNTHESIS:
HYDROGEN TRANSFER
Aimalicine
Zlb-H
I
4
8
A
ppm
FIGURE 2: ‘H NMR spectra (in part) of ajmalicine and [18,20*H,]ajmalicine in deuterioacetone at 400 MHz (imp. = impurities from solvent).
described above (except that BH4- was replaced by NADPH), allowed a comparison of the course of the chemical and enzymatic reduction of cathenamine. If both mechanisms, Le., the chemical and enzymatic reduction, proceed in the same way, one would expect that one D+ would appear in the (2-20 position of each of the ajmalicine isomers. We, therefore, incubated strictosidine in the presence of DzO, NADPH, and the crude C. roseus enzymes. Ajmalicine, 19-epiajmalicine, and tetrahydroalstonine extracted from these reactions were then analyzed by M S and ‘H N M R for their deuterium content. For all three isomers, the M S data were consistent with incorporation of two deuterium atoms, mlz 354, 353, and 339 instead of mlz 352, 351, and 337, respectively, for the unlabeled species. The fragment ion at mlz 184 clearly excluded a labeling at C-21 (Heinstein et al., 1980). The shift of the fragment ion from mlz 223 to mlz 225 indicated an uptake of D+ into the C-19 methyl group and into the C-20 position, respectively (Hesse, 1974). Detailed ‘H NMR analyses unambiguously supported this suggestion. The three isomers ajmalicine, 19-epiajmalicine, and tetrahydroalstonine displayed a triplet for the well-separated signal of C- 19 H by coupling of C- 19 H with the C- 19 CH2D group, whereas the unlabeled alkaloids gave rise to a double quartet due to the coupling between C-19 H and C-19 CH3 (Figure 2). Moreover, coupling between C-20 H, C-21a H, and C-15 H, which is found for the unlabeled alkaloids, disappeared completely. Therefore, the 18,2O-dideuterio compounds would show a clean doublet for 21a-H due to the exclusive geminal coupling with the 21p proton, which indeed was observed. As fully expected, the signals for 15-H were also simplified, since no apparent coupling interactions occurred with 20-D (Figure 2). These results clearly demonstrate the chemical and enzymatic reduction of cathenamine to be identical, namely, addition of H+ at C-20 followed by H- transfer from BH4-
VOL. 22, NO. 14, 1983
3451
or NADPH into the C-21 position. This transfer of the hydride ion should also be easily monitored by M S and ‘H N M R and would be expected to clarify whether the enzymatic reduction steps catalyzed by cathenamine reductase(s) take place in a stereospecific manner. The cell-free conversion of strictosidine to 3a-heteroyohimbines, therefore, was carried out in the presence of NADPD, generated during the incubation by alcohol dehydrogenase from NADP+ and [ZH6]ethanol. The MS spectra of the alkaloids formed showed main fragments at m l z 353, 352, and 338, respectively, which is in agreement with one-deuterium incorporation. A major ion, usually observed at mlz 184 for the undeuterated isomers, was shifted to mlz 185, which suggested a transfer of D- exclusively into the C-21 position of the three ajmalicine isomers. The MS data did not, however, allow a distinction to be made between D- incorporation into the C-2la or C-21p position of the ajmalicine isomers. The stereochemistry of this reduction step, however, can be determined with ’H N M R from the disappearance of the corresponding 21a or 21p signal, as well as by the collapse of the geminal coupling of the corresponding proton. No signal was observed for C-21a H in the spectrum of the 21-2H-labeled alkaloids, ajmalicine and tetrahydroalstonine. 2 1/3-H was displayed as a singlet, whereas in the undeuterated isomers, the coupling constant was 11-12 Hz for the 21a/21/3 hydrogens. Both observations are consistent with a complete and stereospecific transfer of D- into the C-21 position. The spectrum of 19-epiajmalicine revealed no signal for C-21 a H. Therefore, in the enzymatic reduction of cathenamine, the reductase(s) transfer(s) the H- of NADPH in a highly stereospecific mode into the C-2 1a position of all three ajmalicine isomers. During the last 4 years, the role of geissoschizine as an “intermediate” in the biogenetic formation of 3a-heteroyohimbines has been widely studied (Scott et al., 1978; Stockigt, 1978; Lee et al., 1979; Stockigt et al., 1980). The methods discussed above, utilized to elucidate the stereochemistry of the reactions of the main pathway, were also used for the final explanation of the precursor role of geissoschizine in the formation of the three ajmalicine isomers. The enzymatic formation of ajmalicine, 19-epiajmalicine, and tetrahydroalstonine from geissoschizine was investigated in the presence of NADP’INADPD. After isolation of the 3a-heteroyohimbine alkaloids, the spectroscopic analysis gave identical results with those obtained in the experiment when strictosidine was used as the precursor. Again, one deuterium was incorporated into the C-21 position, and the stereochemistry of this reduction step was in absolute agreement with the “strictosidine experiment”. These results clearly show that geissoschizine is indeed converted into the ajmalicine isomers by the enzymes of the main pathway after channeling geissoschizine into the appropriate dehydro form (4,2 1-dehydrogeissoschizine) has occurred in a separate side reaction (Figure 1). Geissoschizine is, in contrast to previous assumptions, not a biogenetic intermediate in heteroyohimbine biosynthesis (Battersby & Hall, 1969; Lee et al., 1979) (see also Added in Proof). The findings presented here, obtained with the isotope deuterium,-are summarized in Figure 1 and confirm that a crude enzyme mixture can be successfully used for the investigation of mechanistic aspects, including the stereochemistry involved, of a complete biogenetic pathway. This methodology, therefore, should provide the possibiity to follow the overall enzymatic synthesis of 3a-heteroyohimbine alkaloids in an NMR tube, employing, e.g., 13C-labeledprecursors, to obtain evidence for so far suggested intermediates of the
3452
BIOCHEMISTRY
pathway. This work is presently in progress. Added in Proof The most recent monograph on indole alkaloid biosynthesis (Atta-ur-Rahman & Basha, 1983) does not take this fact in consideration, because of incompletely reviewed literature. Acknowledgments We are grateful to B. Krausch for excellent technical assistance. Our thanks are due to Dr. B. Deus and the members of our cell-culture laboratory for providing us with cell-suspension cultures and to Dr. V. Wray (GBF) for NMR spectra. Geissoschizine was a kind gift of Prof. Winterfeldt (Hannover). Strictosidine was kindly supplied by U. Pfitzner of our laboratory. Helpful discussion and the continuous interest in this work by Prof. M. H. Zenk and Prof. H. G. Floss are acknowledged. J.S. thanks the Deutsche Forschungsgemeinschaft for a Finnigan MAT 44s instrument. Registry No. Tryptamine, 61-54-1; secologanin, 19351-63-4; strictosidine, 20824-29-7; strictosidine aglycon, 85925- 13-9; dialdehyde, 85955-83-5; carbinolamine, 85925- 14-0; 4,2 1-dehydrocorynantheine aldehyde, 85925- 15-1; 20,21-didehydrocorynantheinealdehyde, 85925- 16-2; 4,2 1-dehydrogeissoschizine, 73385-56-5; cathenamine, 63661-74-5; cathenamine iminium form, 74924-24-6; 19-epicathenamine, 73326-87-1; geissoschizine, 439-66-7; 19-epiajmalicine, 25532-45-0; 19-epicathenamine iminium form, 85955-84-6; ajmalicine, 483-04-5; tetrahydroalstonine, 6474-90-4.
References Atta-ur-Rahman & Basha, A. (1983) in Biosynthesis of Indole Alkaloids (Baldwin, J. E., Goodenough, J. B., Halpern, J., & Rowlinson, J. S., Eds.) International Series of Mono-
STOCKIGT ET AL.
graphs on Chemistry, p 5 5 , Clarendon Press, Oxford. Battersby, A. R., & Hall, E. S. (1969) J . Chem. SOC.,Chem. Commun., 7 93. Bradford, M. M. (1976) Anal. Biochem. 72, 248. Heinstein, P., Stiickigt, J., & Zenk, M. H. (1980) Tetrahedron Lett. 21, 141. Hemscheidt, T., & Zenk, M. H. (1980) FEBS Lett. 110, 87. Hesse, M. (1974) Fortschr. Massenspektrometrie 1, 130. Hofle, G., Heinstein, P., Stockigt, J., & Zenk, M. H. (1980) Planta Med. 40, 120. Husson, H.-P., Kan-Fan, C., Sevenet, Th., & Vidal, J.-P. (1977) Tetrahedron Lett., 1889. Lee, S.-L., Hirata, T., & Scott, A. I. (1979) Tetrahedron Lett. 20, 69 1. Lounasmaa, M., & Kan, S.-K. (1980) Tetrahedron Lett. 36, 1607. Pfitzner, A., & Stijckigt, J. (1982) Phytochemistry 21, 1585. Scott, A. I., Lee, S.-L., Hirata, I., & Culver, M. G. (1978) Rev. Latinoam. Quim. 9 , 131. Stockigt, J. (1978) J. Chem. SOC.,Chem. Commun., 1097. Stockigt, J. (1980) in Indole Biogenetically Related Alkaloids (Phillipson, J. D., & Zenk, M. H., Eds.) Academic Press, London and New York. Stiickigt, J., Hofle, G., & Pfitzner, A. (1980) Tetrahedron Lett. 21, 1925. Treimer, J. F., & Zenk, M. H. (1979) FEBS Lett. 97, 159. Zenk, M. H. (1980) J . Nut. Prod. 43, 438. Zenk, M. H., El-Shagi, H., Arens, H., Stockigt, J., Weiler, E. W., & Deus, B. (1977) in Plant Tissue Culture and Its Biotechnological Application (Barz, W., Reinhard, E., & Zenk, M. H., Eds.) Springer, Berlin, Heidelberg, and New York.
